PCR and other amplification methods offer the best option for sensitive, reliable, cheap and easy detection, characterization, quantitation and discrimination of nucleic acid sequences. A few copies of template target nucleic acid can be replicated in vitro or in situ to provide enough product nucleic acid for analysis. However, the end product often has to be separated and purified from the reaction mixture before its identification and characterization is possible, and whilst amplification can be rapid, identification and characterization can be slow and laborious. The characterization and identification of the nucleic acid amplification product represents a considerable bottleneck in the processing of samples particularly in diagnostic applications of amplification methodologies. For this reason quicker and more convenient methods are desirable for amplification product detection and characterization to improve high throughput screening of biological samples.
With respect to amplification product detection, it is known that PCR products, either modified or unmodified, can be identified and characterized spectrophotometrically. However, determination of the amplification product can be difficult or impossible in the presence of modified or unmodified excess primers present in the reaction mixture at completion of amplification. It is necessary to be able to discriminate between these two species (i.e. amplification products and primers) to permit product detection and characterization, on line, at the end of the amplification process.
In view of the above it is known that single stranded nucleic acid sequences complementary to other nucleic acids can be used to `capture`, by hybridization, such other sequences and that these capture sequences can be derivatized to a support. Such a support (hereinafter known as `the support`) could be a magnetizable or non-magnetizable particle or bead, porous or non-porous membrane, microtitre plate, paper or surface or substrate material that may be contacted with a fluid medium.
Techniques currently exist which make use of these principles. It is possible to detect and characterize a PCR product by its denaturation and subsequent `capture` to a complementary single stranded oligonucleotide immobilized to a support. The support is frequently represented by a microtitre plate; the single stranded capture sequences are bound to the wall of the wells of the plate and are used to hybridize to a denatured, single stranded amplification product. Excess primers are removed from the system by washing the bound product in the microtitre plate wells, and the amplification product is subsequently detected and characterized by spectrophotometric means. However these techniques are frequently time consuming and complex and risk introducing contamination and artefacts into the process.
An alternative approach would be to remove excess primers from the amplification reaction mixture subsequent to amplification and determine the amplification product directly in solution spectrophotometrically. This would have the benefit of being a potentially faster simpler and more efficient process for amplification product/primer separation and detection and characterization. In the present invention such benefits and advantages over known prior art procedures are provided by a substrate/support where separation, detection and characterization of amplification products are analyzed spectrophotometrically in solution.
Surprisingly we have observed that a magnetizable solid phase support (MSPS) derivatized with single stranded nucleic acids having random sequences of nucleotide bases can anneal to and thereby capture single stranded nucleic acids as effectively as capture sequences having nucleotide sequences complementary to those to be captured. This discovery is applicable to the capture of all single stranded nucleic acid species including PCR primers. Essentially this discovery indicates that complementarity between single stranded nucleic acid capture sequences and the sequence to be captured is unnecessary to effect the latter's capture. We have surprisingly observed that a mixture of totally random sequences, present as oligonucleotides, derivatized to a support can function as well as specific, complementary capture sequences in the removal of single stranded nucleic acids from solution, including primers from PCR reaction mixtures.